Alkali myosin light chain (MLC1/3), Alpha-tropomyosin (Alpha-TM) and troponin T (TNT) are three genes expressed in sarcomeric muscles. Each one of these genes generates multiple mRNAs, each coding for a different protein isoform, by a process of alternative splicing. In the case of the MLC1/3 gene, two alternatively spliced mRNAs (coding for MLC1 and MLC3, respectively) are produced from two different and overlapping transcriptional units whose promoters are located 10kb apart but terminate at the same poly A addition site. The Alpha-TM gene produces a minimum of three difference mRNAs that are alternatively spliced at both the 5' and 3' ends. It is possible that the conformation of primary transcript with differences at either the 5' end, the 3' end or both, is responsible for the splice site selection that determines the alternative splicing pathways. Troponin T on the other hand, produces a minimum of 10 different mRNAs, with the potential to generate up to 64, with identical 5' and 3' ends by alternative splicing of internal exons from a unique primary transcript. Since the expression of these different TNT mRNA isoforms is developmental and tissue-specific, it is likely that cell-specific diffusable factors are involved in the regulation of the splicing pathways of this single primary transcript. The specific aims of this proposal are three fold: 1) To determine the relative role of the structure of primary transcripts and putative trans-acting factors in the production of regulated alternative splicing pattern in muscle cells. 2) Using specific gene constructs, to determine the cell and/or gene-specificity of the trans-acting factors involved in alternative splicing by studying their expression in muscle and non-muscle cells. 3) To initiate the characterization of these factors using in vitro and in vivo splicing systems.